This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

A complex of three cell adhesion molecules (CAMs) Neurexin IV(Nrx IV), Contactin (Cont)
and Neuroglian (Nrg) is implicated in the formation of septate junctions between epithelial
cells in Drosophila. These CAMs are interdependent for their localization at septate junctions and e.g.
null mutation of nrx IV or cont induces the mislocalization of Nrg to the baso-lateral membrane. These mutations also
result in ultrastructural alteration of the strands of septate junctions and breakdown
of the paracellular barrier. Varicose (Vari) and Coracle (Cora), that both interact
with the cytoplasmic tail of Nrx IV, are scaffolding molecules required for the formation
of septate junctions.

Results

We conducted photobleaching experiments on whole living Drosophila embryos to analyze the membrane mobility of CAMs at septate junctions between epithelial
cells. We show that GFP-tagged Nrg and Nrx IV molecules exhibit very stable association
with septate junctions in wild-type embryos. Nrg-GFP is mislocalized to the baso-lateral
membrane in nrx IV or cont null mutant embryos, and displays increased mobile fraction. Similarly, Nrx IV-GFP
becomes distributed to the baso-lateral membrane in null mutants of vari and cora, and its mobile fraction is strongly increased. The loss of Vari, a MAGUK protein
that interacts with the cytoplasmic tail of Nrx IV, has a stronger effect than the
null mutation of nrx IV on the lateral mobility of Nrg-GFP.

Conclusion

The strands of septate junctions display a stable behavior in vivo that may be correlated with their role of paracellular barrier. The membrane mobility
of CAMs is strongly limited when they take part to the multimolecular complex forming
septate junctions. This restricted lateral diffusion of CAMs depends on both adhesive
interactions and clustering by scaffolding molecules. The lateral mobility of CAMs
is strongly increased in embryos presenting alteration of septate junctions. The stronger
effect of vari by comparison with nrx IV null mutation supports the hypothesis that this scaffolding molecule may cross-link
different types of CAMs and play a crucial role in stabilizing the strands of septate
junctions.

Background

In insects, septate junctions localize to the apico-lateral domain of epithelial cells
underneath the zonula adherens and play the role of the vertebrate tight junctions
by preventing paracellular diffusion [1]. Septate junctions are also found between the glial cells that isolate the brain
and peripheral nerves and are required for blood-nerve barrier formation in Drosophila [2]. The machinery providing axonal insulation has been conserved for a part during evolution
since, in vertebrate myelinated axons, septate-like junctions attach the terminal
myelin loops to the axonal membrane at paranodes. Both the ultrastructural feature
and the molecular composition of Drosophila septate junctions and vertebrate paranodal junctions are highly conserved [3,4]. Septate junctions are formed by strands of regularly spaced inter-membrane particles.
A complex of CAMs including Nrx IV, Cont and Nrg is critically involved in the organization
of septate junctions in Drosophila whereas the homologous molecules, Caspr/Paranodin, Contactin and Neurofascin-155 are
required for the formation of paranodal junctions in vertebrates [5].

Nrx IV is a transmembrane molecule with a multimodular ectodomain including several
laminin-G and EGF-like domains. Nrx IV interacts in cis with Cont, a glypiated CAM
of the Ig superfamily (Ig-CAM), which only displays heterophilic binding activity
[6,7]. Nrg is a homophilic transmembrane Ig-CAM of the L1 family [8,9] that associates with Cont and Nrx IV at septate junctions. The cytoplasmic tail of
Nrx IV contains a binding site for Coracle (Cora), a member of the Four-point-one,
Ezrin, Radixin, Moesin (FERM) family [10]. It also includes a C-ter PDZ-binding motif interacting with the membrane associated
guanylate kinase (MAGUK) Varicose (Vari) [11].

In Drosophila, null mutation of each of the nrx IV, cont or nrg genes induces embryonic lethality with breakdown of the trans-epithelial and blood-nerve
barriers and alteration of the septate junctions [6,12,13]. In epithelial cells of nrx IV null mutant, Nrg is misdistributed to the baso-lateral membrane and Cont is diffusely
distributed in the cytoplasm indicating that Nrx IV is required for the proper membrane
expression of Cont. Reciprocally in cont null mutant, Nrx IV and Nrg are mislocalized to the baso-lateral membrane. Null mutation
for cora or vari coding for scaffolding molecules of septate junctions cause barrier defects and mislocalization
of Nrx IV. In addition, the PDZ-containing proteins Discs large (Dlg) and Scribble
localized at septate junctions, also control the establishment of epithelial cell
polarity [18]. Several other membrane molecules have been identified as critical components of
the fly septate junctions, including Gliotactin [14], Lachesin [15] and the claudin-related molecules Megatrachea [16] and Sinuous [17]. The loss of one of the septate junction membrane components induces alteration of
the trans-epithelial barrier and mislocalization of the Nrx IV complex at the baso-lateral
region although the network of interactions bridging together these molecules is still
unknown.

An interesting question is whether the strands of septate junctions display either
dynamic characteristics or stable behavior to achieve their role of barrier. Drosophila offers a model system to investigate the membrane mobility of CAMs when recruited
into highly ordered multimolecular complexes at septate junctions of epithelial cells
and when these complexes are disrupted in different genetic backgrounds. We addressed
this question using Fluorescence Recovery After Photobleaching (FRAP) analysis in
live embryos.

Results and discussion

The lateral mobility of Nrg is restricted at septate junctions of epithelial cells
in whole living embryos

We used a Nrg-GFP Flytrap line where a chimera between Nrg and GFP is expressed from the endogenous
Nrg promoter as described in Morin et al. [19] and reflects the tissue and subcellular distribution of the endogenous protein [20,21]. GFP is inserted in the cytoplasmic tail close to the C-ter of Nrg. The homozygous
line is viable and fertile without any apparent phenotype.

We analyzed the lateral mobility of Nrg using FRAP in whole living embryos at stage
15. At this stage, Nrg-GFP expression is restricted to the apico-lateral domain in
epithelial cells indicating it is recruited at septate junctions. A photobleach was
applied in areas of septate junctions and the fluorescence recovery was monitored
in the bleached spot. These experiments were usually not extended for longer time
than 160 s because of extensive epidermis movements. Photobleaching of a 3–5 μm2 area was performed either in the apico-lateral (Fig. 1) or horizontal (Fig. 2) orientation. In either orientation, the recovery of fluorescence in the bleached
area was low in wild-type embryos, and the mobile fraction of Nrg-GFP estimated to
25 % within 160 s (Fig. 3A). We did not observe any difference between the ventral and dorsal epidermis. We
also examined the long-term recovery of Nrg-GFP after photobleaching and only a partial
recovery (39 %) occurred even after 40 min [see Additional file 1]. Thus, our data indicates that Nrg-GFP is strongly stabilized at the apico-lateral
membrane by forming an adhesion complex at septate junctions of wild-type embryos.
As a matter of comparison, a similar stability was reported for the structural components
of the adherens junctions, E-cadherin, α-catenin and Armadillo as analyzed by FRAP
in live Drosophila embryos [22].

Additional file 1. Long term recovery of Nrg-GFP after photobleaching in epithelial cells of live embryos.
Face-on views of epithelial cells in wild-type embryos. Pre-bleach image with the
bleached area indicated with box. Note the partial recovery of fluorescence even until
41 min after bleaching. Bar: 5 μm.

Figure 1.FRAP of GFP-tagged Nrg in epithelial cells of live embryos. Side views of epithelial cells in wild-type (A), nrx IV (B), cont (C) and Dfvari (D) embryos at stage15. Pre-bleach images are shown on the left. The sections of cell
interface that were bleached are indicated with boxes. Images on the right show the
recovery of fluorescence 160 seconds after bleaching. Bar: 5 μm. Schematic representation
of the distribution of septate junction components, Nrg, Nrx IV and Cont in each genotype.

Figure 2.FRAP of Nrg-GFP in embryonic epithelial cells. Face-on views of epithelial cells in wild-type (A), nrx IV (B), cont (C) and Dfvari (D) embryos at stage15. Pre-bleach images are shown on the left. The sections of cell
interface that were bleached are indicated with boxes. Images on the right show the
recovery of fluorescence 160 seconds after bleaching. Bar: 5 μm.

In nrx IV null mutant embryos, Nrg-GFP mislocalized to the baso-lateral membrane of epithelial
cells and its mobile fraction strongly increased (44 % within 160 s) by comparison
with wild-type embryos (Fig. 1B, 2B and 3B). The high mobility of Nrg-GFP in nrx IV mutant embryos may be correlated with disruption of the septate junction tripartite
complex. It must be noted that Cont is diffusely distributed in the cytoplasm of epithelial
cells in nrx IV mutants [6] implicating that both Nrx IV and Cont are lacking at the epithelial cell membrane.
The loss of nrx IV is accompanied by an almost complete lack of junctional strands as analyzed by electron
microscopy [12].

Similarly, in cont mutants, Nrg-GFP was expressed at the baso-lateral membrane of epithelial cells and
the mobile fraction of Nrg-GFP (40 % within the time of 160 s) strongly increased
when compared with wild type embryos (Fig. 1C, 2C and 3C). In cont or nrx IV mutant embryos, Nrg does not take part into the tripartite adhesion complex of septate
junctions but may still be engaged in homophilic binding between two neighboring epithelial
cells [23,24]. Under these last conditions, exchange between free and bound Nrg-GFP receptors may
occur rapidly allowing lateral diffusion along the plasma membrane (Fig. 4B).

Figure 4.Organization of adhesion complexes at epithelial cell contacts in the wild-type and
mutant embryos. (A) Molecular organization of septate junctions in wild-type embryos. NrxIV forms
a tripartite complex with Cont and Nrg. A highly ordered complex may be generated
due to the binding of Nrx IV with the scaffolding molecules Vari and Cora. Several
additional components including Gliotactin, Sinuous and Dlg are required for the formation
of septate junctions, but the network of interactions mediating bridging with the
Nrx IV complex is largely unknown. Vari, which displays a multimodular structure,
may homo-multimerize and mediate linkage with Cora, Dlg or Sinuous. FRAP analysis
indicates that the mobile fraction of Nrg and Nrx IV is low. (B) In nrx IV mutant embryos, the tripartite complex is missing, Cont is not targeted to the cell
membrane and Nrg may bind homophilically. The mobile fraction of Nrg is increased
to 45 % indicating rapid exchange between bound and unbound molecules. However, the
presence of Vari and Cora may limit for a part the lateral mobility of Nrg by cross-linking
with other components of septate junctions. (C) In cora or vari null mutant embryos, the tripartite complex between Nrx IV, Cont and Nrg may still
occur, but the network of interactions mediated by the scaffolding molecules is disrupted.
The mobile fractions of Nrx IV and Nrg are increased up to 64–70 %.

The restricted mobility of Nrg and Nrx IV at septate junctions depends on the scaffolding
components Vari and Cora

We used a Nrx IV-GFP Flytrap line where GFP is inserted into the first intron between
the N-ter signal sequence and the discoïdin domain [25]. This line is homozygous viable and fertile and Nrx IV-GFP expression is restricted
to the apico-lateral domain in epithelial cells of stage 12–15 embryos indicating
it is recruited at septate junctions as the endogenous protein (Fig. 5A). We performed FRAP analysis on wild-type embryos at stage 15 and determined that
the mobile fraction of Nrx IV-GFP was 27 % within 160 s in septate junctions of epithelial
cells (Fig. 6A).

Figure 5.FRAP of Nrx IV-GFP in embryonic epithelial cells. Side (A) and face-on (B) views of wild-type embryos, side (C) and face-on (D) views
of Dfvari embryos at stage15. Face-on (E) and side (F) views of cora mutant embryos. Pre-bleach images are shown on the left. The bleached areas are indicated
with boxes. Images on the right show the recovery of fluorescence 160 seconds after
bleaching. Bar: 5 μm in A-E and F. Schematic representation of the distribution of
septate junction components, Nrx IV, Vari and Cora in each genotype.

Figure 6.Kinetics of the fluorescence recovery of Nrx IV-GFP. Photobleaching in the horizontal plane of epithelial cells from wild-type (A), Dfvari (B) and cora (C) embryos. Data are normalized to yield an intensity of zero immediately after photobleaching
and represents the average of 9 individual traces (mean ± SEM). The traces were obtained
using FRAP sequences from multiple embryos [wild-type (n = 3), cor (N = 4) and vari (N = 2)]. Statistical analysis using ANOVA indicates that the loss of Vari or Cora
significantly (P < 0.01) increased the mobile fraction of Nrx IV-GFP as compared with
wild-type embryos.

Next, we analyzed the mobility of both Nrg-GFP and Nrx IV-GFP in a deficiency (Dfvari) for vari, which encodes a MAGUK that binds the cytoplasmic tail of Nrx IV [11]. As shown in Fig. 1D and 5C, the loss of vari induces the mislocalization of both Nrg-GFP and Nrx IV-GFP at the baso-lateral membrane.
In this deficiency, the recovery fraction of Nrg-GFP after photobleaching is very
high (70 % within the time of 160 s) (Fig. 1D, 2D and 3D). Similarly, the mobile fraction of Nrx IV-GFP (65 % within the time of 160 s) strongly
increased by comparison with wild-type embryos (Fig. 5C–D and 6B). The MAGUK protein Vari that contains L27, PDZ, SH3, HOOK, and GUK modules may act
as a scaffolding molecule at septate junctions. The PDZ domain of Vari binds Nrx IV
and its HOOK domain may be implicated in homo-multimerization or anchoring Dlg, another
MAGUK associated with septate junctions [26] and interacting with Gliotactin [14]. Indeed, mutations in the HOOK domain impair the accumulation of Vari at septate
junctions [11]. Therefore Vari may support oligomerization of transmembrane components of the septate
junction complex or induce linkage with submembrane elements (Fig. 4A).

It must be noted that the mobile fraction of Nrg-GFP is significantly increased in
Dfvari embryos by comparison with nrx IV null mutant embryos (70 % instead of 45 %). This is in accordance with genetic evidences
indicating that Vari may display functions beyond interacting with Nrx IV and bridge
together several elements of the septate junctions. As a matter of the fact, vari mutations can strongly enhance the phenotype caused by mutations of the claudin sinuous, whereas nrx IV mutations do not [17].

Finally, the mobility of Nrx IV-GFP was analyzed in a null allele for cora (Fig. 5E). Nrx IV-GFP localized at the baso-lateral membrane in the mutant embryos (Fig. 5F). As observed in Dfvari embryos, the mobile fraction of Nrx IV-GFP (64 % within the time of 160 s) strongly
increased by comparison with wild-type embryos (Fig. 6C). The FERM domain of Cora is required for the recruitment of Nrx IV at septate junctions
[10]. However, how Cora would generate a scaffold is unknown since it does not contain
an actin-spectrin binding domain like its related vertebrate homologue 4.1 adaptors.
Our data suggest that Cora may participate to the septate junction scaffold beyond
interacting with Nrx IV since its mutation has a similar effect as the loss of Vari.

Lateral mobility of Caspr/Paranodin in transfected cells

Next, we compared the dynamics of the Nrx IV CAM when anchored or not at septate junctions
in live embryos with the mobility of its vertebrate orthologue Caspr/Paranodin at
the membrane of transfected cells without any junctional complex or intercellular
contacts. We examined the lateral mobility of Paranodin fused with GFP at the C-ter
in neurobastoma N2a cells. Paranodin-GFP was co-transfected together with Contactin,
which is required for its cell surface expression in vertebrate cells [27]. In this context, cell-cell contacts do not induce the formation of septate-like
junctions as observed at the level of axo-glial paranodal junctions. FRAP experiments
were performed by bleaching a section of cell membrane (Fig. 7A). The mean mobile fraction of Paranodin-GFP was 47 % within the time of 160 s (Fig.
7B).

Figure 7.Role of the cytoplasmic region in the lateral mobility of paranodin analyzed by FRAP. (A) Images of neuroblastoma N2a cells co-transfected with paranodin-Δcyto (pndΔcyto)
and contactin showing an example of a FRAP sequence. Bar: 5 μm. Kinetics of fluorescence
recovery of GFP-tagged paranodin (B) and paranodinΔcyto (C). Data are normalized to
yield an intensity of zero immediately after photobleaching and represents the average
of 8 individual traces (mean ± SEM). Deletion of the cytoplasmic tail of paranodin
significantly (P < 0.05) increased the mobile fraction of paranodin-GFP as assessed
using ANOVA.

We evaluated the role of the cytoplasmic region of Paranodin in its lateral mobility
at the cell membrane. We analyzed the mobility of a mutant form, ParanodinΔcyto, with
deletion of the cytoplasmic tail. The length of the C-ter of transmembrane proteins
has been shown to influence their lateral mobility [28] since a minimal length should be required for the restriction by dynamic barriers.
In the Paranodin-GFP constructs, GFP (265 aa) is fused at the C-ter and is expected
to minimize the size effect of the cytoplasmic tail deletion (73 aa). This deletion
mutant displayed a mobile fraction of 60 % (Fig. 7C), which is significantly enhanced by comparison with full-length Paranodin. Paranodin
contains a juxta-membrane motif for the binding of 4.1 molecules, which are adaptors
for linkage with the actin-spectrin cytoskeleton. Paranodin interacts with 4.1B at
the paranodal junctions [29] similarly to Nrx IV that binds Cora in Drosophila septate junctions [10]. However, in contrast to Nrx IV, Paranodin does not contain a C-ter PDZ-binding domain.
When co-transfected in Cos cells, Paranodin forming a complex with Contactin interacts
with the endogenously expressed 4.1 molecule Schwannomin and deletion of the 4.1-binding
motif prevents this interaction [30]. Thus, the association of the cytoplasmic tail of Paranodin with a 4.1 scaffolding
molecule may reduce its lateral mobility.

Conclusion

We report here the first in vivo measurements of the dynamics of septate junctions, which display similar stability
as adherens junctions in Drosophila epithelial cells. Septate junctions form a barrier
to trans-epithelial diffusion but may also act as a fence within the plane of the
plasma membrane to prevent diffusion of membrane-bound proteins between the apical
and basolateral membrane domains. However, septate junctions that are fully assembled
at late stages of the embryogenesis do not play a critical role in the maintenance
of membrane asymmetry in fly epithelial cells although they could play a redundant
function together with adherens junctions [18]. In vertebrates, the septate-like junctions at paranodes play such a role of fence
by limiting the lateral diffusion of the voltage-gated sodium and potassium channels
along the axon [31]. Our data indicating that membrane proteins are stably anchored at the septate junctions
are compatible with such a function of lateral barrier.

Our in vivo FRAP analyses indicate that the Nrg and Nrx IV molecules inserted in the strands of
septate junctions are quite stable likely in terms of the arrangement of CAMs within
strands and behavior of the strands. As observed for CAMs within septate junctions,
claudin molecules are not mobile within paired strands as examined in artificially
induced tight junction-like structures within apposing membranes of fibroblasts transfected
with GFP-claudin-1 [32]. However, the network of claudin strands show a very dynamic behavior although this
observation is not necessarily representative of tight junction strands in epithelial
cells, because of the lack of ability of the GFP-claudin-1 to bind PDZ domain-containing
proteins such as ZO-1, and then, to be cross-linked with the underlying cytoskeleton.

The restricted mobility of CAMs at septate junctions depends on both adhesive interactions
and clustering by scaffolding molecules. The membrane mobility of Nrg-GFP and Nrx
IV-GFP is strongly increased in mutant embryos presenting alteration of septate junctions.
An important distinction between the effects of the loss of cell membrane and scaffolding
components has been revealed by dynamic analysis. Mutations of septate junction CAMs
significantly increase the mobility of the transmembrane components, but mutations
in scaffolding elements have an even greater effect. These data underline the major
role of scaffolding molecules in stabilizing highly ordered adhesion complexes at
septate junctions.

Methods

Drosophila stocks

Stocks were obtained from the Bloomington Drosophila Stock Center and published sources
contex956 [6], nrx IV4304 [12] and cora1 [33]alleles have been described previously. The deficiency Dfvari (w1118; Df(2L)Exel7079, P+PBac{XP5.WH5}Exel7079/CyO) was used as a null mutant for vari [11]. The Flytrap lines Nrg-GFP (G00305) [19] and Nrx IV-GFP (CA06597) [25] obtained from the FlyTrap project contain the GFP exon inserted in the nrg and nrx IV genes, respectively. New mutant lines were generated with the following phenotypes:
nrg-GFP; contex956/TM3, nrg-GFP; nrx IV4304/TM3, nrg-GFP; Dfvari/CyO; cora1/CyO; nrx IV-GFP, Dfvari/CyO; nrx IV-GFP.

Preparation of embryos

Embryos were collected on yeast agar plates and aged to stage 15 (12 h–13 h 30 at
25°C). The embryos were dechorionated for 2 min with bleach (6% chlore), rinsed with
water, blotted dry with paper towels, gently picked up with double-sided tape and
placed in a Petri dish and covered with water for FRAP analysis.

Transfected cells and Caspr/Paranodin constructs

The DNA construct pRc-CMV/F3 encodes the full-length sequence of F3/Contactin, Paranodin-GFP
encodes the full-length sequence and Paranodin-Δcyto encodes the extracellular and
transmembrane domains of Caspr/Paranodin (aa1-1297) fused with GFP in pEGFP-N1 [34]. Neuroblastoma N2a cells plated on coverslips were grown in DMEM containing 10% FCS
and transiently transfected using jet PEI (Ozyme, Saint Quentin Yvelines, France).
The coverslips were placed in a Petri dish in a temperature controlled chamber at
37°C for FRAP analysis, 48-h after transfection.

FRAP analysis

The FRAP analysis was performed on a Leica (Wetzlar, Germany) TCS SP2 laser scanning
microscope equipped with 1.2 NA 63× water immersion objective. Imaging of GFP was
performed using the 488-nm beam of an argon laser at 7% laser power, with the electronic
zoom at 5×, scan speed of 400 Hz, box size 512 × 512 pixels. Images (two averages)
were acquired with an open pinhole. These conditions were found to give minimal photobleaching
over the observed time. The ROI for bleaching (3–5 μm2) was delineated and the FRAP sequence was started: 3–6 reference images were acquired
first, then the sample was bleached by the laser at 100 % for 3 s (2 bleach scans),
and fluorescence recovery was recorded for 160 s using the Leica FRAP software. Acquisition
of z-stacks before and after bleaching indicates that the bleached spot can be detected
over 2.8 μm in the z-axis [see Additional file 2]. Thus, even in case of epidermal movements in the z-axis, images of the recovery
sequence are acquired within the bleached volume. In addition, all the images of the
FRAP sequences were checked by eye to correct the ROI position for epidermal movements
in xy. To create the recovery curves, the background-corrected fluorescence intensities
were transformed into a 0–1 scale and plotted using Microsoft Excel. The individual
traces were fitted using SigmaPlot through the data of the function Im*(1-exp(-t/τ)) to calculate the mobile fraction Im, and the recovery time τ.

Additional file 2. Bleaching of Nrx IV-GFP along the z-axis in epithelial cells of live embryos. Confocal
xy- and z-sections (stacks of 8 sections, 400 nm step) of epithelial cells in wild-type
embryos. Pre-bleach and post-bleach images with the bleached area indicated with box.
Quantification of fluorescence intensities into the bleached area of confocal sections.
Bar: 5 μm.

Multiple embryos (2–5) for each genotype were analyzed. FRAP experiments were conducted
in the same sample on heterozygous and homozygous embryos for each of the nrx, cont, cor, and vari mutations. Similar kinetics of fluorescence recovery were obtained for Nrg-GFP or
Nrx IV-GFP in wild-type and heterozygous embryos.

Authors' contributions

ML participated in the design of the study, carried out the fly genetic crossing,
participated to the FRAP analysis and helped to draft the manuscript. CB carried out
the FRAP analysis in neuroblastoma cells and performed the statistical analysis. CF–S
conceived the study, performed FRAP analysis on Drosophila embryos and drafted the
manuscript. All authors read and approved the final manuscript.

Acknowledgements

We are grateful to the Bloomington stock center and to the FlyTrap project. We wish
to thank M-P Blanchard for assistance with confocal and FRAP imaging. This work was
supported by the Agence Nationale de la Recherche and the National Multiple Sclerosis
Society.

References

Tepass U, Hartenstein V: The development of cellular junctions in the Drosophila embryo.